Embryonically modified animal and method of constructing the same

ABSTRACT

The present invention relates to a method for efficiently producing a reproducible animal using totipotent cells wherein mitochondrial DNAs (e.g., wild-type DNAs) adapted to nuclear DNAs have been introduced into or substituted with mitochondrial DNAs, and the present invention also relates to an animal obtained by such production method. When the totipotent cells are ES cells derived from an inbred mouse, the tetraploid rescue method is preferably used. In the production of chimeric animals, mitochondrial DNAs of totipotent cells derived from an animal to be used is substituted with wild-type mitochondrial DNAs by the back-crossing method, the nuclear replacement method, or the like, and the cells are injected into a tetraploid fertilized egg, so that a reproducible inbred chimeric animal is produced while avoiding death of the obtained inbred chimeric animal from respiratory disturbances and the like immediately after birth. The thus obtained reproducible chimeric animal can be used for gene function analyses, animal experiments, and the like without carrying out complicated manipulations for generating inbred animals.

TECHNICAL FIELD

The present invention relates to a method for efficiently producing areproducible animal derived from manipulated embryo that shows germ-linetransmission when gene-altered animals such as knockout mice areproduced. More specifically, when totipotent cells such as embryonicstem cells (ES cells) are produced, the present invention relates to amethod for producing an animal derived from totipotent cells that isprepared by back-crossing, cell fusion, mitochondria injection,pronuclei replacement, somatic nuclear transfer, or the like tointroduce or substitute a desired type of mitochondrial DNAs.Particularly when an animal species is a mouse, the present inventionrelates to a method for efficiently producing reproducible inbredgerm-line chimeric mice by improving the birth rate and the survivalrate of newborns.

The present invention further relates to a reproducible mouse that isproduced by any one of the above methods, wherein mitochondrial DNAs areadapted type mitocondrial DNAs that are of a genotype by which nonuclear-mitochondrial incompatibility takes place, and the totipotentcells are transmitted to the germ line.

BACKGROUND ART

Conventionally, technology exists for producing chimeric mice precedingthe technology for production of so-called gene-targeted mice such asknockout mice. Chimeric mice are produced using ES cells that have beenproven to be transmitted to a germ line. As such ES cells proven to betransmitted to a germ line, TT2 cells of an ES cell line establishedfrom hybrids of C57BL/6 mice and CBA mice and ES cell lines establishedfrom mice 129 substrains, which are non-standard strains (there are 3standard strains of mouse: BALB/c, C57BL/6, and C3H), are used. Hence,to use chimeric mice in phenotype analyses or as experimental animals ingene function analyses and the like, there is a need to introduce targetgene alteration in the produced mice (gene-altered mice) into inbredmice by a back-crossing method. Specifically, complicated proceduresthat have been required include producing gene-altered ES cells using EScells of a hybrid strain or non-standard strains, producing germ-linechimeric mice using the cells, and then repeatedly back-crossing thethus obtained hetero-type gene-altered mice (generally, male mice areused) with inbred mice of a standard strain (e.g., C57BL/6), so as togenerate inbred mice of the standard strain.

In contrast, direct production of germ-line chimeric mice of an inbredstrain has been attempted by establishing ES cells from inbred mice.However, although there have been reports that germ-line chimeric micecould be obtained from ES cells derived from inbred mice such as C57BL/6above, this has not yet been practically applied. Currently, hybridstrains are used as described above. Therefore, it is substantiallydifficult to obtain ES cells of good inbred mice to obtain germ-linechimeric mice.

In addition, it is said that ontogenesis using only ES cells issubstantially impossible.

In the meantime, a tetraploid rescue method has been devised as a methodfor selectively producing chimeric mice from ES cells (Nagy A et al.,Development 110, 815-821 (1990)). This method is based on the knowledgethat tetraploid cells develop into placenta, and only ES cells developinto an individual body when they are injected into tetraploidfertilized eggs. This technique is called the tetraploid rescue method.However, according to a recent report concerning comparison andexamination conducted for the tetraploid rescue method and nucleartransfer technology, most newborns died because of respiratorydisturbances after birth and only one inbred chimeric mouse could beproduced and obtained by the tetraploid rescue method, when ES cellsestablished from BALB/c mice were used (Eggan K et al., PNAS 98,6209-6214 (2001)). Furthermore, also regarding clone mouse productionusing somatic nuclear transfer technology, it has been reported thatmost newborns of the obtained inbred mice died after birth because ofrespiratory disturbances, and viable newborn clone mice could beobtained through the use of cell nuclei of hybrid mice (Wakayama T etal., Nature 394, 369-374 (1998); Wakayama T et al., PNAS 96, 1484-1498(1999); Humpherys D et al., Science 296, 95-97 (2001)). As describedabove, even if no apparent abnormalities are observed, chimeric micegenerated by the tetraploid rescue method only from ES cells of inbredmice and clone mice derived from somatic cell nuclei of inbred mice diein many cases because of respiratory disturbances. Such problem isinferred to take place when an animal species is changed to mammalsother than mice.

Moreover, successfully produced conventional knockout mice are germ-linechimeras obtained using ES cells derived from mice of a hybrid strain ormice 129 substrains, non-standard strains, but are not inbred mice.Reports have been extremely rare that when ES cells derived from inbredmice were used, chimeric mice could be produced. None has been reportedsubsequently. No germ-line transmission has been reported in mostsuccessful cases, and it has been substantially impossible to obtaingerm-line chimeric mice. As described above, the tetraploid rescuemethod has been thought to be the sole means to address the problemsaccompanying inbred chimeric mouse production. However, most newbornsobtained by this method die immediately after birth because ofrespiratory disturbances (Eggan K et al., PNAS 98, 6209-6214 (2001)).Thus, it has been impossible to actually obtain reproducible chimericmice.

SUMMARY OF THE INVENTION

Under the above circumstances, we have intensively studied theproduction of animals derived from manipulated embryo such asreproducible chimeric animals. In particular, we have studied theproduction of a chimeric mouse, wherein mitochondrial DNAs are adaptedtype (the mitochondrial DNAs, by which no nuclear-mitochondrialincompatibility, such as wild-type mitochondrial DNAs, takes place) andtotipotent cells are transmitted to a germ line.

Specifically, an object of the present invention is to efficientlyobtain animals derived from manipulated embryos such as reproduciblechimeric animals, in particular, inbred chimeric animals, and furtherparticularly, inbred chimeric mouse individuals. Particularly, an objectof the present invention is to develop a method for producing: inbredchimeric animals derived from totipotent cells such as ES cells derivedfrom inbred animals used in production of chimeric animals obtained bythe tetraploid rescue method; and inbred chimeric animals andparticularly inbred chimeric mice, while avoiding death of the inbredchimeric mice immediately after birth because of respiratorydisturbances.

Specifically, the object of the present invention is, in production ofchimeric animals by the tetraploid rescue method, to provide a methodfor efficiently producing: inbred chimeric animals derived fromtotipotent cells such as ES cells derived from inbred animals used; andreproducible inbred chimeric animals and particularly inbred chimericmice, while particularly avoiding death of inbred chimeric miceimmediately after birth because of respiratory disturbances.

A second object of the present invention is to provide reproducibleinbred chimeric mice that are animals derived from manipulated embryo,such as chimeric animals obtained by the above production method, whoserate of death immediately after birth because of respiratorydisturbances is particularly low, and wherein mitochondrial DNAs areadapted type mitochondrial DNAs and totipotent cells are transmitted toa germ line.

Furthermore, a third object of the present invention relates tototipotent cells and particularly embryonic stem cells that are used forproducing such animal derived from a manipulated embryo.

Totipotent cells in the present invention mean undifferentiated cellscapable of differentiating into any type of cell. Examples of such cellsinclude embryonic stem cells and nuclear transferred embryos.

Characteristics of the present invention relate to: a method forproducing the above-described reproducible animals derived frommanipulated embryo and particularly reproducible germ-line chimericanimals; reproducible chimeric animals, and particularly chimeric miceand reproducible inbred chimeric mice produced by such method; andtotipotent cells used for the production.

Specifically, the present invention is a method that can be applied foranimals derived from manipulated embryos, and particularly chimeric miceobtained as a result of production of gene-altered mice such asso-called knockout mice. The method is characterized in that it involvesintroducing adapted type mitochondrial DNAs into a cell to be subjectedto injection or substituting mitochondrial DNAs of the cell to besubjected to injection with adapted type mitochondrial DNAs, or using acell wherein mitochondrial DNAs of a host embryo has been introducedtherein or substituted with adapted type mitochondrial DNAs. In thiscase, totipotent cells having adapted type mitochondrial DNAs introducedtherein and an untreated host embryo, untreated totipotent cells and ahost embryo having adapted type mitochondrial DNAs introduced therein,totipotent cells having adapted type mitochondrial DNAs introducedtherein and a host embryo having adapted type mitochondrial DNAsintroduced therein, or the like can be used. The thus produced chimericanimal has the adapted type mitochondrial DNAs. Examples of such adaptedtype mitochondrial DNAs include wild-type-derived mitochondrial DNAs,and a Mus musculus musculus-type DNAs of a wild-type mouse. Whengerm-line chimeric animals derived from totipotent cells such as EScells produced from inbred animals and particularly chimeric micecompletely derived from ES cells are produced, totipotent cells such asES cells of an inbred mouse wherein mitochondrial DNAs have beenintroduced or substituted is used as cells to be injected into atetraploid fertilized egg. In this manner, death of the obtained inbredchimeric mice immediately after birth because of respiratorydisturbances and the like can be avoided, and reproducible inbredchimeric mice can be efficiently produced.

To date, when inbred chimeric mice selectively derived from ES cells areproduced by the tetraploid rescue method using the ES cells of inbredmice, most of the obtained chimeric mice died immediately after birthbecause of respiratory disturbances. It has actually been impossible toobtain reproducible chimeric mice by the tetraploid rescue method. Theexistence of abnormal DNA methylation patterns has been posed as areason why most newborns obtained by the tetraploid rescue method diebecause of respiratory disturbances (Dean W et al., Development 125,2237-2282(1998)). In addition to this reason, we were able to confirmfor the first time that incompatibility between mitochondrial DNAs andnuclear DNAs is also a reason. Hence, we could have avoided deathimmediately after birth by converting mitochondrial DNAs of ES cellsderived from an inbred mouse into mitochondrial DNAs of a wild-typemouse for the DNAs able to be adapted to nuclear DNAs. When we appliedthis technology to inbred animals (mice), we could efficiently producegerm-line chimeric mice. We believe that we can expect a similar effectalso in the case of clone animals and general diploid chimeric animalsif adapted type mitochondrial DNAs are introduced into animals that areproduced by embryo manipulation.

The present invention provides the following methods, an animal derivedfrom a manipulated embryo and particularly a chimeric animal produced bysuch methods, and totipotent cells.

-   1. A method for producing an animal derived from manipulated embryo    derived from totipotent cells, comprising using cells having adapted    type mitochondrial DNAs introduced therein which adapt to nuclear    DNAs.-   2. The method for producing an animal of 1 above, wherein the animal    derived from the manipulated embryo derived from the totipotent    cells is a chimeric animal.-   3. The method for producing an animal of 2 above, wherein the    chimeric animal is an inbred chimeric animal.-   4. The method for producing an animal of any one of 1 to 3 above,    comprising using totipotent cells having adapted type mitochondrial    DNAs introduced therein and an untreated host embryo, untreated    totipotent cells and a host embryo having adapted type mitochondrial    DNAs introduced therein, or totipotent cells having adapted type    mitochondrial DNAs introduced therein and a host embryo having    adapted type mitochondrial DNAs introduced therein.-   5. The method for producing an animal of 4 above, comprising using a    tetraploid fertilized egg as a host embryo for an animal and    totipotent cells as cells having adapted type mitochondrial DNAs    introduced therein.-   6. The method for producing an animal of 5 above according to a    tetraploid rescue method, comprising injecting the totipotent cells    having the adapted type mitochondrial DNAs introduced therein into    the blastocyst of a tetraploid fertilized egg.-   7. The method for producing an animal of any one of 1 to 6 above,    wherein the cells having the adapted type mitochondrial DNAs    introduced therein are cells which have the adapted type    mitochondrial DNAs as a result of substitution.-   8. A method for producing a clone individual by nuclear transfer    using a recipient oocyte having adapted type mitochondrial DNAs that    adapt to nuclear DNAs of a donor cell.-   9. The method for producing an animal of any one of 1 to 8 above,    wherein nuclear-mitochondrial incompatibility results in respiratory    disturbances immediately after birth.-   10. The method for producing an animal of any one of 1 to 9 above,    wherein the adapted type mitochondrial DNAs are mitochondrial DNAs    derived from a wild type.-   11. The production method of any one of 1 to 10 above, wherein the    animal is a mouse.-   12. The method for producing an animal of any one of 1 to 11 above,    wherein the method for substituting mitochondrial DNAs with desired    mitochondrial DNAs is either a back-crossing method or a pronuclei    replacement method.-   13. The method for producing an animal of any one of 9 to 12 above,    wherein the desired mitochondrial DNAs are of a wild-type mouse Mus    musculus musculus type.-   14. A reproducible chimeric animal, which is produced by any one of    the methods of 1 to 13 above, and wherein mitochondrial DNAs are    introduced into or substituted with adapted type DNAs, and    totipotent cells can be transmitted to a germ line.-   15. The animal of 14 above, wherein the animal is a mouse.-   16. A totipotent cell for producing a reproducible genetically    manipulated animal, wherein mitochondrial DNAs have been introduced    into or substituted with adapted type DNAs, and its character is    transmitted to a germ line.-   17. The totipotent cell of 16 above, wherein adaptation with nuclear    DNAs cause a decrease in respiratory disturbances immediately after    birth.-   18. The totipotent cell of 16 or 17 above, wherein adapted type    mitochondrial DNAs have been introduced as mitochondrial DNAs.-   19. The totipotent cell of 18 above, wherein the adapted type    mitochondrial DNAs are of a wild-type mouse Mus musculus musculus    type.-   20. The embryonic stem cell of any one of 16 to 19 above, wherein    the genetically manipulated animal is a mouse.

BEST MODE OF CARRYING OUT THE INVENTION

It is preferable to obtain an animal derived from a manipulated embryoobtained by the method of the present invention through the use of atetraploid rescue method using totipotent cells such as ES cells or thenuclear transfer embryo of an inbred animal. Hence, the animals obtainedaccording to the present invention are basically inbred animals whereinmost cells including germ-line cells are derived from totipotent cellsof inbred animals used. Furthermore, since the mitochondrial DNAs oftotipotent cells such as ES cells of an inbred animal to be used havebeen previously substituted with those of an animal of a wild-typespecies by pronuclei replacement or the like, the obtained chimericanimal grows without dying from respiratory disturbances immediatelyafter birth. Therefore, reproducible chimeric animals can be efficientlyobtained, and inbred animals that can be utilized for gene functionanalyses or as experimental animals can be directly obtained from theobtained germ-line chimeric animals. Hence, this is very useful becausethere is no need to repeat time-consuming and complicated crossing togenerate inbred animals. Such animals may be any animals as long as theyare animals regarding which it has been reported that chimeras can begenerally produced therewith. Inbred animals are particularlypreferable. Such animals may preferably be mammals, further preferablyrodents, and particularly preferably mice.

Totipotent cells such as ES cells derived from these animals areprepared by a known method, and then used in the present invention.Embodiments of the use of ES cells derived from a mouse are explained asfollows. It is naturally understood that the animal species used in thepresent invention are not particularly limited to a mouse.

A mouse-derived mitochondrial DNAs-substituted ES cells are prepared byintracellular substitution of mitochondrial DNAs according to the aboveconventional technology. Substitution of mitochondrial DNAs of an inbredmouse is conducted by producing an inbred mouse (in many cases, theinbred mouse is of a standard strain such as C57BL/6, C3H, or BALB/c)having mitochondrial DNAs of a desired type as a result of substitutionby the back-crossing method, the nuclear transfer method, or the like,and then establishing ES cells from the mouse, so as to be able toobtain ES cells having the substituted mitochondrial DNAs to be used inthe present invention. When C57BL/6 inbred mice are repeatedly crossedwith Mus musculus musculus wild-type mice by the back-crossing method 12times or more, B6-mtMus inbred mice wherein the mitochondrial DNAs ofthe C57BL/6 inbred mice have been substituted with those of Mus musculusmusculus type of the wild-type mouse can be produced. From the mice, EScells wherein the mitochondrial DNAs have been substituted with the Musmusculus musculus-type DNAs of an adapted type mouse can be established.

In the meantime, when the pronuclei replacement method is employed,according to the standard method (McGrath J & Solter D, J. Exp. Zool.228, 355-362 (1983)), male and female pronuclei of the pronuclear-stagefertilized egg of an mdx inbred mouse are transplanted into anenucleated pronuclear-stage fertilized egg having a Mus musculusmusculus-type mitochondria of the wild-type mouse. Nuclear fusion iscarried out by electric pulses, the egg is cultured, and then the egg istransplanted into a mouse oviduct for ontogenesis, so that an mdx-mtMusinbred mouse wherein the mitochondrial DNAs have been substituted withthose of the Mus musculus musculus type can be produced. Similarly, EScells wherein the mitochondrial DNAs have been substituted with those ofa wild type can be established from this mouse.

To establish ES cells, a blastocyst-stage embryo is collected from theinbred mouse produced in this manner, such as B6-mtMus or mdx-mtMus, andthen ES cells can be established by a standard method (Evans M J andKaufman M K, Nature 292, 154-156 (1981)).

ES cells having a target character obtained by the above method isinjected into an embryo of a fertilized egg, preferably a tetraploidembryo that has divided into a blastocyst-stage tetraploid fertilizedegg, thereby producing a chimeric mouse.

Blastocyst-stage tetraploid embryos can be obtained by naturally matingICR mice that had been subjected to superovulation induction treatmentwith male mice of the same strain, collecting late 2-cell stage embryosby flushing the oviduct method from mouse individuals for which plugshave been confirmed, conducting fusion treatment by the electric pulsemethod to prepare tetraploid embryos, and then culturing the embryos inM16 media or CZB media. ES cells prepared from the prescribed B6-mtMusor mdx-mtMus inbred mice are injected into the obtained blastocyst-stageembryos using a microinjector such as a piezomicromanipulator. Next, thethus obtained blastocyst-stage embryos having had the ES cells injectedtherein are transplanted into the uteri of recipient ICR mice aftercrossing and 2.5 days after plug confirmation. On the night beforedelivery (night of day 18.5 of pregnancy), chimeric mouse fetuses aretaken out by Cesarean section, and then resuscitated. After theconfirmation of active movement of the fetuses, they are raised bypreviously prepared foster parents. Most of the thus obtained newborns(chimeric mice) conducted spontaneous respiration, and grew normallywith almost no malformation. The chimeric mice that had grown hereinwere characterized in that all cells, including germ line cells, werederived from the ES cells, and in that such mice can reproduce with atarget animal and proliferate.

EXAMPLES

The present invention will be hereafter described in detail by referringto examples. However, the examples are intended to merely illustrate theinvention, and the invention is not limited by these examples.

Example 1 Production of Inbred Mice Having Substituted MitochondrialDNAs

(1) Production of Inbred Strains B6-mtMus and B6-GFP-mtMus Having MusMusculus Musculus-Type Mitochondrial DNAs Substituted by theBack-Crossing Method

Female mice (8-week-old) were obtained by crossing 8-week-old maleC-57BL/6J inbred mice with 8-week-old female Mus musculus musculuswild-type mice. Back-crossing between the obtained female mice, again,8-week-old male C57BL/6J inbred mice was repeatedly carried out. Byrepeating crossing 12 times or more, B6-mtMus inbred mice were producedwherein mitochondrial DNAs have been substituted with those of Musmusculus musculus type. In addition, the mitochondrial DNAs wereanalyzed by the Nested-PCR method (Kaneda H et al., PNAS 92, 4542-4546(1995)), so that it was confirmed that the DNAs had been substitutedwith those of Mus musculus musculus type.

Furthermore, female B6-mtMus (N18) mice were crossed with male B6-GFP#4mice (Ichida et al.), thereby producing B6-GFP-mtMus mice.

(2) Production of Inbred Disease Model Mice C57BL/10J-mdx-mtMus(mdx-mtMus) by Pronuclei Replacement Method

A 3-week-old female mdx inbred disease model mouse (C57BL/10-mdx) and a3-week-old female hybrid mice (hybrid mice (CBF1-mtMus) resulting from♀BALB-mtMus×♂C57BL/6) having Mus musculus musculus-type mitochondrialDNAs of a 3-week-old wild-type mouse were subjected to superovulationinduction treatment (pregnant mare serum gonadotropin (PMSG) wasadministered at the rate of 5 IU/0.05 ml/mouse, and 48 hours later humanchorionic gonadotropin (hCG) was administered at the rate of 5 IU/0.05ml/mouse). The female hybrid mice were allowed to live together with8-week-old male ICR mice for natural mating. At 24 hours after hCGadministration, eggs were collected so as to prepare pronuclear-stagefertilized eggs. Using the thus obtained pronuclear-stage fertilizedeggs, pronuclear replacement was conducted according to a standardmethod (McGrath J & Solter D, J. Exp. Zool. 228, 355-362 (1983)).Specifically, male and female pronuclei of the pronuclear-stagefertilized eggs, the donor fertilized eggs derived from the aboveC57BL/10-mdx inbred disease model mice, were introduced into enucleatedpronuclear-stage fertilized eggs, the recipient fertilized eggs derivedfrom the above CBF1-mtMus hybrid mice having the Mus musculusmusculus-type mitochondrial DNAs of the wild-type mouse. After beingsubjected to nuclear fusion by electric pulse, the eggs were culturedovernight in an M16 medium supplemented with 0.1 mM EDTA (94.59 mM NaCl,4.78 mM KCl, 1.71 mM CaCl₂, 1.19 mM KH₂PO₄, 1.19 mM MgSO₄, 25.07 mMNaHCO₃, 23.28 mM sodium lactate, 0.33 mM sodium pyruvate, 1 g/L glucose,4 g/L BSA, 100 IU/mL penicillin, and 50 μg/mL streptomycin) underconditions of 37° C. and 5% CO₂-5% O₂-90% N₂, and then transplanted inmouse oviducts. A total of 16 eggs were transplanted (8 eggs peroviduct) into the oviducts of female ICR mice on day 0.5 ofpseudo-pregnancy (The female mice were crossed with vasoligated malemice, on the next day, plug confirmation was conducted, and the noon ofthis day was determined to be day 0.5). As a result, 3 female and 2 malepups were obtained. The 3 thus obtained female mice (8-week-old orolder) as founders were allowed to live together with male mdx(C57BL/10-mdx) mice, thereby obtaining 2nd generation mice. Similarly,2nd generation female mice were crossed with male mdx mice, therebyobtaining 3rd generation mice. The thus obtained 3rd generationmitochondrial DNAs were verified by the PCR-RFLP method. Common primerswere designed within a D-loop (primer production referring to Kaneda Het al., PNAS 92, 4542-4546 (1995): 5′-AATTATTTTCCCCAAGC-3′ (SEQ ID NO: 1in Sequence Listing) and 5′-AGAAGAGGGGCATTG-3′ (SEQ ID NO: 2 in SequenceListing)). A 262-bp fragment obtained by amplification was treated withDra1 restriction enzyme, and then unfragmented Mus musculus domesticustype DNAs and Mus musculus musculus type DNAs fragmented to a 122-bpfragment and a 140-bp fragment were verified. It was confirmed that themitochondrial DNAs of the thus obtained 3rd generation mice had beensubstituted with those of Mus musculus musculus type, thereby producinginbred disease model mice C57BL/10J-mdx-mtMus (mdx-mtMus).

Example 2 Production of ES Cells from Inbred Mice Having Substituted MusMusculus Musculus-Type Mitochondrial DNAs

The inbred female B6-mtMus (N15) mice having the substituted Musmusculus musculus-type mitochondrial DNAs obtained in Example 1 (1) werenaturally crossed with male C57BL/6 mice. The uterus of each mouseindividual was perfused with an M2 medium on day 3 after plugconfirmation, and then 20 blastocyst-stage embryos were collected. Usingthe obtained embryos, ES cells were established according to a standardmethod (Evans M J and Kaufman M K, Nature 292, 154-156 (1981)).Specifically, the previously obtained embryos were transferred one byone onto feeder cells in a 4-well plate to which an ES medium (80%(v/v)DMEM, 20% (w/v) FCS, 1% (v/v) 100 mM pyruvate solution (Gibco Cat.11360-070), 1% (v/v) 100× nonessential amino acid solution (Gibco Cat.11140-027), 1 mM 2-mercaptoethanol (Sigma, Cat. No. M-6250), and 10³U/mL LIF had been apportioned. The embryos were then cultured underconditions of 37° C. and 5% CO₂-5% O₂-90% N₂. On day 5 of culture, ICMcell aggregation that had grown was selected. 0.125% trypsin/0.1 mM EDTAtreatment was conducted to disperse the cells, and then the cells wereseeded and cultured on feeder cells newly prepared in a 4-well plate. Atthe 2nd passage, only ES-cell-like colonies were selected, and thenseeded and cultured on a new 4-well plate by the method same as thatemployed in the 1st time. At the 3rd passage, entire wells were treatedwith trypsin/EDTA, and then the cells were seeded on a new 3.5 cmculture plate, thereby obtaining 2 lines that were able to grow well.The cells that could reach this stage could then also stably grow, sothat the cells were cryopreserved as a cell line.

Furthermore, 4 lines of ES cells were established by conducting similarmanipulations using 6 blastocyst-stage embryos obtained from the inbreddisease model mice mdx-mtMus obtained in Example 1 (2) and having thesubstituted Mus musculus musculus-type mitochondrial DNAs.

When the mitochondrial DNAs of the ES cells established from the above 2mouse strains were tested, they were confirmed to be of Mus musculusmusculus type.

Example 3 Preparation of Tetraploid Blastocyst-Stage Embryo

ICR mice subjected to superovulation induction treatment were naturallycrossed with male mice of the same strain. Late 2-cell stage embryoswere collected by the tubal perfusion method from mouse individuals forwhich plugs had been confirmed at 44 to 46 hours after theadministration of human chorionic gonadotropin (hCG). The thus obtainedlate 2-cell stage embryos were subjected to fusion treatment by directcurrent electric pulse in 0.3 M mannitol (Goku manufactured by FUJIHIRA,and pulse conditions of 18 V, at intervals of 50 μsec and 999 μsec (3times each)), thereby preparing tetraploid embryos. The tetraploidembryos were selected, and then cultured in a M16 medium at 37° C. in 5%CO₂-Air for two days, thereby obtaining tetraploid blastocyst-stageembryos.

Example 4 Production of Chimeric Mice by Tetraploid Rescue Method (1)

ES cells (gender: male) prepared from the prescribed B6-mtMus inbredmice were injected using a piezomicromanipulator into the tetraploidblastocyst-stage embryos obtained in Example 3. Approximately 10 to 15ES cells were injected into a tetraploid blastocyst-stage embryo. 76tetraploid blastocyst-stage embryos were subjected to injection. 71tetraploid blastocyst-stage embryos into which the ES cells had beensuccessfully injected were implanted into the uteri of 4 recipient ICRmice on day 2.5 after plug confirmation. On the night before delivery(night of day 18.5 of pregnancy), chimeric mouse fetuses were taken outby Cesarean section, and then resuscitated. After active movement of thefetuses was confirmed, they were raised by previously prepared fostermothers. The 13 thus obtained male newborns showed no malformation andconducted spontaneous respiration, and 4 of these mice grew normally.

Example 5 Production of Chimeric Mice by Tetraploid Rescue Method (2)

ES cells (gender: male) prepared from the prescribed mdx-mtMus inbreddisease model mice were injected using a piezomicromanipulator into thetetraploid blastocyst-stage embryos obtained in Example 3. Approximately10 to 15 ES cells were injected into each tetraploid blastocyst-stageembryo. 108 tetraploid blastocyst-stage embryos were subjected toinjection. 89 tetraploid blastocyst-stage embryos into which the EScells had been successfully injected were transplanted into the uteri of5 recipient ICR mice on day 2.5 after plug confirmation. On the nightbefore delivery (night of day 18.5 of pregnancy), fetuses were taken outby Cesarean section, and then resuscitated. After active movement of thefetuses was confirmed, they were raised by previously prepared fosterparents. The 19 thus obtained male newborns showed almost nomalformation and conducted spontaneous respiration, and 11 of these micegrew normally.

Example 6 Production of Chimeric Mice by Tetraploid Rescue Method (3)

ES cells (gender: female) prepared from the prescribed B6-GFP-mtMusinbred mice were injected using a piezomicromanipulator into thetetraploid blastocyst-stage embryos obtained in Example 3. Approximately10 to 15 ES cells were injected into each tetraploid blastocyst-stageembryo. 75 tetraploid blastocyst-stage embryos were subjected toinjection. 75 tetraploid blastocyst-stage embryos to which the ES cellshad been successfully injected were implanted in the uteri of 3recipient ICR mice on day 2.5 after plug confirmation. On the nightbefore delivery (night of day 18.5 of pregnancy), chimeric mouse fetuseswere taken out by Cesarean section, and then resuscitated. After activemovement of the fetuses was confirmed, they were raised by previouslyprepared foster parents. 8 out of 12 female newborns showed nomalformation and conducted spontaneous respiration. 7 mice died becauseof child neglect or were killed by recipient mice. The remaining 1 mousepups grew normally.

Example 7 Confirmation of Proliferation of Chimeric Mice Produced byTetraploid Rescue Method

Two male chimeric mice obtained from ES cells prepared from the inbreddisease model mdx-mtMus obtained in Example 1 were crossed with 2 normalfemale mdx-mice, thereby obtaining 38 newborns. These mice showed nomalformation and grew smoothly. Furthermore, each of all the mouseindividuals showed typical features of mdx (C57BL/10-mdx) inbred diseasemodel mice. Thus, it could be confirmed that the chimeric mice obtainedby the method of the present invention can leave normal pups in futuregenerations.

INDUSTRIAL APPLICABILITY

Implementation of the present invention enables reproducible chimericanimals, particularly inbred chimeric animals, and further particularlyinbred chimeric mouse individuals to be efficiently obtained. Inparticular, the implementation of the present invention enables toefficiently produce inbred animals while avoiding death because ofrespiratory disturbances immediately after the birth of inbred animalsand particularly chimeric mice derived from totipotent cells such as EScells or nuclear transferred embryos derived from inbred animals used inproducing chimeric animals obtained by the tetraploid rescue method. Thethus obtained chimeric mouse is a reproducible inbred mouse which hasmitochondrial DNAs adapted to nuclear DNAs and wherein totipotent cellsare transmitted to a germ line.

1. A method for producing an animal derived from manipulated embryoderived from totipotent cells, comprising using cells having adaptedtype mitochondrial DNAs introduced therein which adapt to nuclear DNAs.2. The method for producing an animal of claim 1, wherein the animalderived from the manipulated embryo derived from the totipotent cells isa chimeric animal.
 3. The method for producing an animal of claim 2,wherein the chimeric animal is an inbred chimeric animal.
 4. The methodfor producing a chimeric animal of any one of claims 1 to 3, comprisingusing totipotent cells having adapted type mitochondrial DNAs introducedtherein and an untreated host embryo, an untreated totipotent cells anda host embryo having adapted type mitochondrial DNAs introduced therein,or totipotent cells having adapted type mitochondrial DNAs introducedtherein and a host embryo having adapted type mitochondrial DNAsintroduced therein.
 5. The method for producing an animal of claim 4,comprising using a tetraploid fertilized egg as a host embryo for ananimal and totipotent cells as cells having adapted type mitochondrialDNAs introduced therein.
 6. The method for producing an animal of claim5 according to a tetraploid rescue method, comprising injecting thetotipotent cells having the adapted type mitochondrial DNAs introducedtherein into the blastocyst of a tetraploid fertilized egg.
 7. Themethod for producing a chimeric animal of any one of claims 1 to 6,wherein the cells having the adapted type mitochondrial DNAs introducedtherein is cells which have the adapted type mitochondrial DNAs as aresult of substitution.
 8. A method for producing a clone individual bynuclear transfer using a recipient oocyte having adapted typemitochondrial DNAs that adapt to a nuclear DNAs of a donor cell.
 9. Themethod for producing an animal of any one of claims 1 to 8, whereinnuclear-mitochondrial incompatibility results in respiratorydisturbances immediately after birth.
 10. The method for producing ananimal of any one of claims 1 to 9, wherein the adapted typemitochondrial DNAs are mitochondrial DNAs derived from a wild type. 11.The production method of any one of claims 1 to 10, wherein the animalis a mouse.
 12. The method for producing an animal of any one of claims1 to 11, wherein the method for substituting mitochondrial DNAs withdesired mitochondrial DNAs is either a back-crossing method or apronuclei replacement method.
 13. The method for producing an animal ofany one of claims 9 to 12, wherein the desired mitochondrial DNAs are ofa wild-type mouse Mus musculus musculus type.
 14. A reproduciblechimeric animal, which is produced by any one of the methods of claims 1to 13, and wherein mitochondrial DNAs are introduced into or substitutedwith adapted type DNAs, and totipotent cells can be transmitted to agerm line.
 15. The animal of claim 14, wherein the animal is a mouse.16. A totipotent cell for producing a reproducible geneticallymanipulated animal, wherein mitochondrial DNAs are of adapted type, andits character is transmitted to a germ line.
 17. The totipotent cell ofclaim 16, wherein adaptation with nuclear DNAs causes a decrease inrespiratory disturbances immediately after birth.
 18. The totipotentcell of claim 16 or 17, wherein adapted type mitochondrial DNAs havebeen introduced as mitochondrial DNAs.
 19. The totipotent cell of claim18, wherein the adapted type mitochondrial DNAs are of a wild-type mouseMus musculus musculus type.
 20. The embryonic stem cell of any one ofclaims 16 to 19, wherein the genetically manipulated animal is a mouse.